The aberrantly changed level of homocysteine (Hcy) triggers a variety of pathological symptoms and subsequently Hcy-related diseases. Direct and selective visualization of Hcy in biological systems is pivotal to understanding the pathological functions of Hcy at the molecular level. Herein, a general strategy was developed for the specific fluorescence imaging of Hcy through the combination of dual-binding sites and the introduction of a nitro group at the 6-position of the 7-diethylaminocoumarin fluorophore. Also, a series of novel fluorescent probes were exploited for monitoring Hcy with excellent selectivity, high sensitivity, and far-red/near-infrared fluorescence emission. Furthermore, fluorescence imaging of endogenous Hcy dynamics in living cells and in vivo was achieved, providing direct and solid evidence for the increasement of endogenous Hcy in type 2 diabetes mellitus and Alzheimer's disease. This research will greatly advance the development and understanding of the molecular nexus between the Hcy metabolism cascade and the root causes of diseases related to Hcy.